GFP

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(GFP as a measure of gene expression)
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* Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
* Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
* A206K: make monomeric
* A206K: make monomeric
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==GFP as a measure of gene expression==
 
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<biblio>
 
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#Lindow pmid=11698362
 
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#Iafolla pmid=18350571
 
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#Bagh pmid=18352063
 
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#Dong pmid=18352052
 
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</biblio>
 
==References==
==References==
 +
 +
===General===
<biblio>
<biblio>
#prasher pmid=1347277
#prasher pmid=1347277
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#zacharias [http://www.tsienlab.ucsd.edu/Publications/Zacharias%202006%20-%20Molecular%20Biology%20and%20Mutation%20of%20GFP.pdf Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)]
#zacharias [http://www.tsienlab.ucsd.edu/Publications/Zacharias%202006%20-%20Molecular%20Biology%20and%20Mutation%20of%20GFP.pdf Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)]
#shaner05 pmid=16299475
#shaner05 pmid=16299475
 +
</biblio>
 +
 +
===GFP as a measure of gene expression===
 +
<biblio>
 +
#Lindow pmid=11698362
 +
#Iafolla pmid=18350571
 +
#Bagh pmid=18352063
 +
#Dong pmid=18352052
</biblio>
</biblio>

Revision as of 22:41, 30 July 2008

Contents

General Info

  • From jellyfish Aeqorea victoria
  • wtGFP sequence on NCBI
  • 238 amino acids
  • The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical [1].
  • Oxygen is required for maturation of the fluorophore

GFP Variants

  • GFPmut3b - S65G, S72A
  • GFPmut3* - mutations from wt: S2R,S65G,S72A
    • This is GFPmut3b[2] with an accidental mutation at position 2 that doesn't affect function according to the authors.[3]
  • Emerald - F64L, S65T, S72A, N149K, M153T, I167T
  • EGFP - added GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [4]
  • Other variants and their mutations can be found in Shaner supplementary table 2 [5]

Mutations

  • F64L: improve folding @37C
  • S65T: 5-6x increase in amplitude and red shift
  • S65(G|T) and T203(Y|F|W|H) -> YFP
  • Y66W: GFP -> CFP
  • Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
  • Y66F: excitation 360nm, emission 442nm
  • R96A: slows cyclization reaction from minutes to months
  • Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
  • A206K: make monomeric

References

General

  1. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien) [zacharias]
  2. Cormack BP, Valdivia RH, and Falkow S. . pmid:8707053. PubMed HubMed [Cormack]
  3. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. . pmid:9603842. PubMed HubMed [Andersen]
  4. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. . pmid:9353317. PubMed HubMed [li97]
  5. Shaner NC, Steinbach PA, and Tsien RY. . pmid:16299475. PubMed HubMed [shaner05]
  6. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, and Cormier MJ. . pmid:1347277. PubMed HubMed [prasher]
    original cloning of GFP

  7. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. . pmid:8303295. PubMed HubMed [chalfie]
    original use of GFP as a reporter

  8. Tsien RY. . pmid:9759496. PubMed HubMed [tsien98]
    good review of GFP

All Medline abstracts: PubMed HubMed

GFP as a measure of gene expression

  1. Leveau JH and Lindow SE. . pmid:11698362. PubMed HubMed [Lindow]
  2. Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, and McMillen DR. . pmid:18350571. PubMed HubMed [Iafolla]
  3. Bagh S, Mazumder M, Velauthapillai T, Sardana V, Dong GQ, Movva AB, Lim LH, and McMillen DR. . pmid:18352063. PubMed HubMed [Bagh]
  4. Dong GQ and McMillen DR. . pmid:18352052. PubMed HubMed [Dong]
All Medline abstracts: PubMed HubMed
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